Expression of glycoprotein D of herpes simplex virus

ABSTRACT

Methods and compositions are provided for the efficient production in yeast of polypeptides which are immunologically cross-reactive with glycoprotein D of the herpes simplex virus. Synthetic DNA fragments encoding for a portion of the glycoprotein, and the naturally-occurring gene encoding for the glycoprotein and portions thereof, are expressed on plasmids in yeast. Secretion may be provided by including a secretory leader and signal processing sequence derived from the α-factor gene. Alternatively, the genes may be expressed intracellularly under the transcriptional control of a promoter derived from a gene in the yeast glycolytic pathway. 
     E. coli strains HB101 containing plasmids pYHS109 and pYHS118 were deposited at the American Type Culture Collection on July 11, 1984, and granted accession nos. 39762 and 39763, respectively.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The herpesviruses include the herpes simplex viruses, comprising twoclosely related variants designated types 1 (HSV-1) and 2 (HSV-2). HSV-1and HSV-2 are responsible for a variety of human diseases, such as skininfections, genital herpes, viral encephalitis, and the like.

The herpes simplex virus is a double stranded DNA virus having a genomeof about 150 to 160 kbp packaged within an icosahedral nucleocapsidenveloped in a membrane. The membrane includes a number ofvirus-specific glycoproteins, the most abundant of which are gB, gC, gDand gE, where gB and gD are cross-reactive between types 1 and 2.

It is a matter of great medical and scientific interest to provide safeand effective vaccines against both HSV-1 and HSV-2. One promisingapproach has been the use of isolated glycoproteins which have beenshown to provide protection when injected into mice subsequentlychallenged with live virus. One limitation on the use of such "subunit"vaccines, however, has been the difficulty in obtaining sufficientamounts of the glycoproteins. Heretofore, such glycoproteins have beenobtained primarily from membranes isolated from a viral culture. Theproblems associated with large scale culturing of these pathogens andthe difficulty in isolating the glycoproteins from the viral genome havesubstantially precluded the use of glycoprotein vaccines.

It would therefore be desirable to provide a safe and efficient methodfor the large-scale production of herpes simplex glycoproteins suitablefor use as vaccines. In particular, it would be desirable to provide forthe production of a polypeptide having an amino acid sequence similar toglycoprotein D, or a portion thereof, which polypeptide can elicit animmune response against both HSV-1 and HSV-2.

2. Description of the Prior Art

Subunit vaccines extracted from chick embryo cells infected with HSV-1or HSV-2 are described in U.S. Pat. Nos. 4,317,811 and 4,374,127. Seealso, Hilfenhaus et al. (1982) Develop. Biol. Standard 52: 321-331,where the preparation of a subunit vaccine from a particular HSV-1strain (BW3) is described. Roizman et al. (1982) Develop. Biol. Standard52: 287-304, describe the preparation of non-virulent HSV-1×HSV-2recombinants and deletion mutants which are shown to be effective inimmunizing mice. Watson et al. (1982) Science 218: 381-384 describe thecloning and low level expression of the HSV-1 gD gene in E. coli, aswell as expression of a cloned fragment by injection into the nuclei offrog oocytes. They also present the nucleotide sequence for the gD gene.Weis et al. (1983) Nature 302: 72-74 report higher level expression ofgD in E. coli. This polypeptide elicits neutralizing antibodies inrabbits. Berman et al. (1983) Science 222: 524- 527 report theexpression of glycoprotein D in mammalian cell culture. Lasky et al.,Biotechnology, June 1984, pp. 527-532 report the use of thisglycoprotein D for the immunization of mice. Cohen et al. (1984) J.Virol. 49: 120-108 reports the localization and chemical synthesis of aparticular antigenic determinant of gD, contained within residues 8-23of the mature protein.

SUMMARY OF THE INVENTION

Novel methods and DNA constructs are provided for the production ofpolypeptides which are immunologically cross-reactive with glycoproteinD (gD) of the herpes simplex virus (HSV). Production of gD in a yeasthost provides the advantages of high levels of expression andmodification of the polypeptides not available with prokaryotic hosts,without necessitating the use of problematic techniques required formammalian cell culture. The polypeptide products are useful as vaccines,as reagents for the immunological detection of the virus, for theproduction of antibodies and monoclonal antibodies, for the detection ofantibodies specific for gD, and the like.

The polypeptides produced by the present invention will include at leastabout nine contiguous amino acids defining an epitopic site found innaturally-occurring glycoprotein D, and may include the entireglycoprotein D sequence. A DNA sequence expressing the desiredpolypeptide is incorporated in a yeast expression vector, which is usedto transform a suitable yeast host. Polypeptides having molecularweights up to about 30 to 50 kilodaltons may include a secretory leaderand processing signal sequence providing for secretion of the cleavedpolypeptide into the nutrient medium. The polypeptide may then becollected from the nutrient medium without having to lyse the yeasthosts. Alternatively, the polypeptides may be expressed intracellularly,preferably employing a promoter derived from an enzyme in the yeastglycolytic pathway, e.g., glyceraldehyde phosphate dehydrogenase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the construction of plasmids pYHS109 and pYHS110 whichcarry synthetic sequences gD-A and gD-B, respectively, as described inthe Experimental section hereinafter.

FIG. 2 is a partial restriction map of the gD region which notes thelocation of all the gD sequences inserted into yeast expression vectors,as described in the Experimental Section hereinafter.

FIG. 3 represents the construction of plasmid pYHS115 which carries thenaturally-occurring gD gene of HSV-1 under the transcriptional controlof the GAPDH promoter and terminator, as described in the Experimentalsection hereinafter.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Polypeptides which are immunologically cross-reactive withnaturally-occurring glycoprotein D are produced in yeast by recombinantDNA methodology. Production in yeast allows the advantages associatedwith eukaryotic hosts, e.g., post-translational modification andsecretion, without the problems associated with maintaining a mammaliancell culture. The polypeptides of the present invention may be producedfrom relatively short synthetic DNA fragments encoding for at leastabout nine amino acids to provide haptens useful for eliciting an immuneresponse specific for gD, e.g., for use in vaccine production. Thecloned DNA fragments (or the RNA produced therefrom) may also find useas hybridization probes in a variety of applications. Alternatively,much longer DNA fragments, either synthetic or natural, may be used toproduce larger polypeptides for use as vaccines, immunogens,immunological reagents, and the like. The peptides of the presentinvention will find particular use in the preparation of vaccinesagainst infection by herpes simplex virus.

The glycoprotein D (gD) DNA fragments of the present invention may be ofnatural or synthetic origins. The natural gD gene of HSV-1 is located onthe viral genome between the short internal repeat (IR_(S)) sequence andshort terminal repeat (TR_(S)) sequence at the 3'-end thereof. Codingfor the mature protein is found on an approximately 1.6 kbp fragmentlocated on a 2.9 kbp SacI restriction fragment of the genome. The entirecoding region for the mature protein is located within a HindIII-NruIfragment of the 2.9 kbp SacI fragment. The naturally-occurring gD genemay be employed with or without modification. Regions of the gene may bedeleted and/or joined to other DNA fragments as desired. Thepreparation, cloning and expression of particular fragments of thenaturally-occurring gD gene are described in detail in the Experimentalsection hereinafter.

Synthetic gD sequences will also find use, either alone or incombination with the naturally-occurring sequences. Coding for thesynthetic sequences may be based on published nucleotide sequences forgD found in the literature. See, Watson et al. (1982), supra. Thesynthetic fragments will be sufficiently long to code for at least ninecontiguous amino acids with a maximum length equal to that of thenaturally-occurring gene or longer. When preparing polypeptides for useas immunological reagents or as vaccines, it is usually desirable thatthe nucleotide sequence code for an oligopeptide corresponding to anepitopic site of the natural gD protein. Often, such epitopic sites maybe predicted by employing the folding rules of Chou and Fasman (1974)Biochemistry 13: 211-222, in conjunction with an analysis of hydrophobicand hydrophilic regions of the protein (Hopp and Woods, 1981, Proc.Natl, Acad. Sci. USA 78: 3824-3828). In particular, it has been foundthat polypeptides corresponding to amino acids 253-283 and 8-23 in themature naturally-occurring gD protein will cross-react with antiseraraised against the natural gD protein.

A number of techniques are available for synthesizing shortsingle-stranded DNA fragments, e.g., the phosphoramidite methoddescribed by Beaucage and Carruthers (1981) Tetrahedron Lett. 22:1859-1862. Short fragments having a length of about 40 bases or less maybe synthesized as a continuous single-stranded fragment. Thecomplementary strand may also be synthesized, and the two strandsannealed under appropriate conditions. Alternatively, the complementarystrand may be added using DNA polymerase with an appropriate primersequence.

The synthesis of longer fragments exceeding 100 base pairs is typicallyaccomplished by the preparation of a series of single-stranded DNAfragments including from about 10 to 100 bases, usually from about 15 to60 bases. The sequences of such fragments are selected so that when thefragments are brought together under annealing conditions, a doublestranded fragment having the desired nucleotide sequence is produced.When devising such synthetic nucleotide sequences, the sequences of theindividual single stranded DNA fragments are examined to assure thatannealing of unmatched segments is avoided. In this way, base pairingoccurs only between those segments which are intended to be annealed inthe resulting double stranded DNA fragment. Undesired base pairing mayusually be avoided by altering one nucleotide (usually the thirdnucleotide) in preselected ones of the codons.

When preparing synthetic gD DNA fragments, it may sometimes be desirableto modify the reported nucleotide sequence. For example, it will usuallybe preferred to use codons which are preferentially recognized by theintended yeast host. Specifically, such codons are those which appear athigh frequency in the structural genes encoding for the yeast glycolyticenzymes. The nucleotide sequence will usually comprise at least 50%preferred yeast condons, more usually at least 60%, and preferably atleast 75%. In some instances, it may be desirable to further alter thenucleotide sequence to create or remove restriction sites or tosubstitute one or more amino acids in the resulting polypeptide. Suchchanges may be made to enhance the immunogenicity of the polypeptide,facilitate conjugating the polypeptide to a carrier protein, or thelike, etc. It may also be desirable at times to add amino acids to theN-terminus or C-terminus of the polypeptide, where such additional aminoacids may provide for a desired result.

The DNA fragments coding for a desired gD fragment will be incorporatedin DNA constructs capable of self-replication and expression in yeast.Such DNA constructs will include a replication system recognized by theyeast host, the gD DNA fragment encoding the desired polypeptideproduct, transcriptional and translational initiation regulatorysequences joined to the 5'-end of the gD sequence, and transcriptionaland translational termination regulatory sequences joined to the 3'-endof the gD sequence.

The transcriptional regulatory sequences will include a promoter, whichmay be the promoter associated with the secretory leader and processingsignal sequence. Other promoters may also find use, particularly thoseinvolved with the enzymes in a yeast glycolytic pathway, such as thepromoters for alcohol dehydrogenase, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), pyruvate kinase, triose phosphate isomerase,phosphoglucoisomerase, phosphofructokinase, and the like. By employingthese promoters with other regulatory sequences, such as enhancers,operators, and the like, and using a host having an intact regulatorysystem, one can regulate the expression of the gD polypeptide product byvarying the carbon source, e.g., replacing glucose with galactose;varying the concentration of a nutrient, e.g., phosphate, or changingthe temperature with a temperature sensitive promotor or regulatorysystem.

The transcriptional termination regulatory sequence will include aterminator, preferably a terminator balanced with the promoter toprovide for proper transcription. Conveniently, the terminator which isnaturally found with the promoter may be employed. The remainingsequences in the construct, including the replication systems for bothyeast, e.g., 2μ plasmid, and bacteria, e.g., Col E1, are well known andamply described in the literature.

Enhanced yields of shorter polypeptides may be obtained by employing DNAconstructs which include a secretory leader and processing signalsequence to effect secretion and post-translational modification of thegene product in yeast. The upper size limit on the secreted polypeptidesis not fixed, although they will usually be below 40 kilodaltons, moreusually below about 30 kilodaltons. Moreover, size is not the onlydeterminative criteria as hydrophobic peptides may be more readilysecreted than non-hydrophobic peptides. The secretory leader andprocessing signal sequences will normally be derived fromnaturally-occurring DNA sequences in yeast which provide for secretionof a polypeptide. Such polypeptides which are naturally secreted byyeast include α-factor, a-factor, acid phosphatase, and the like. Ifdesired, the naturally-occurring sequence may be modified, for example,by reducing the number of lys-arg pairs which define the processing site(while retaining at least one pair), or by reducing the length of thesecretory leader sequence (while retaining sufficient length to providefor secretion), or by introducing point mutations, deletions or othermodifications which facilitate manipulation, e.g., introducingrestriction recognition sites. Conveniently, the secretory leader andprocessing signal sequence may be joined to the gD DNA fragment byproviding appropriate cohesive ends on the gD fragment, by use ofappropriate adaptor molecules, or a combination of both.

Polypeptides of the present invention may also be producedintracellularly as follows. After the transformed cell culture hasreached a high density, the cells will be separated, typically bycentrifugation, lysed, and the gD polypeptides isolated by varioustechniques, such as extraction, affinity chromatography,electrophoresis, dialysis and combinations thereof.

The polypeptides of the present invention, and fragments thereof, may beemployed in a variety of ways. The polypeptides can be employed both aslabelled and unlabelled reagents in various immunoassays, bioassays, andthe like, for the detection of HSV or antibodies to HSV. Suitable labelsinclude radionuclides, enzymes, fluorescers, chemiluminescers, enzymesubstrates or co-factors, enzyme inhibitors, particles, dyes, and thelike. Such labelled reagents may be used in a variety of well knownassays, such as radioimmunoassays, enzyme immunoassays, e.g., ELISA,fluorescent immunoassays, and the like. See, for example, U.S. Pat. Nos.3,766,162; 3,791,932; 3,817,837; 3,996,345; and 4,233,402.

Polypeptides of the present invention may also find use in vaccinesagainst herpes. The larger polypeptides, having a molecular weightexceeding about 5,000 daltons, may be used without further modification.The smaller haptens, however, should be conjugated to an appropriateimmunogenic carrier in order to elicit the desired immune response.Suitable immunogenic carriers include tetanus toxoid. It will also bepossible to link short DNA fragments expressing the gD polypeptides togenes expressing proteins from other pathogenic organisms or viruses. Inthis way, the resulting fused proteins may provide immunity for morethan one disease. In preparing a vaccine, the polypeptides will normallybe incorporated in a physiologically acceptable medium, such as water,saline, phosphate buffered saline, and the like. The vaccine may beadministered intravenously, intraarterially, subcutaneously,intraperitoneally, or the like. The amount of immunogen employed perdose will be about 5 to 10 μg, if liquid, in a volume of about 0.25 to 1ml, and may be administered repeatedly at two to four week intervals,usually not more than two to three times.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

All DNA manipulations were done according to standard procedures. SeeMolecular Cloning, T. Maniatis et al., Cold Spring Harbor Lab., 1982.Enzymes used in cloning were obtained either from New England Biolabs orBethesda Research Laboratories and employed according to the supplier'sdirections. Yeast were transformed and grown using a variety of mediaincluding selective medium (yeast nitrogen base without leucine); YEPDmedium, containing 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v)glucose, and others as appropriate and/or detailed below. In the case ofplating medium contained 2% (w/v) agar and for transformation 3% topagar.

I. Construction of yeast expression vectors containing synthetic DNAfragments coding for polypeptides A and B of the gD gene.

Nucleotide sequences designated gD-A and gD-B based on portions of theamino acid sequence for glycoprotein D of HSV-1 reported by Watson etal. (1982) Science 218: 381-384, and employing preferred yeast condons,were devised. The gD-A sequence, which encodes amino acids 253-283(corresponding to amino acids 258-288, as incorrectly numbered by Watsonet al., supra.) of the mature protein, was as follows: ##STR1##

The gD-B sequence, which encodes amino acids 8-23 (corresponding toamino acids 13-28, as incorrectly numbered by Watson et al., supra.) ofthe mature protein, was as follows: ##STR2##

The sequences each include a KpnI cohesive end at the 5'-end and a SalIcohesive end at the 3'-end. Coding for the mature secreted peptidebegins after the LysArg processing site. The 5'-end of each sequence isa modification of the 3'-end of the naturally-occurring α-factorsecretory leader and processing signal sequence, where the modificationconsists of a deletion of three glu-ala pairs and a replacement of aleucine by a proline to create a KpnI site in the nucleotide sequence.The 3'-end of each sequence includes two translational stop codons, OPand OC.

Synthetic DNA fragments having the sequences just described wereprepared by synthesizing overlapping ssDNA segments as described byUrdea et al. (1984) Proc. Natl. Acad. Sci. USA 80: 7461-7465 using thephosphoramidite method of Beaucage and Caruthers (1981) TetrahedronLett. 27: 1859-1862, and annealing and ligating under the followingconditions.

The ssDNA fragments were joined as follows: 50 to 100 pmoles of eachsegment (except the two segments at the 5'-terminii) were5'-phosphorylated with 5.6 units of T4 polynucleotide kinase (NewEngland Nuclear) in 10 mM dithiothreitol (DDT), 1 mM ATP, 10 mM MgCl₂,100 ng/ml spermidine, 50 mM Tris-HCl, pH 7.8 (total volume: 20 μl) for30 min at 37° C. Additional T4 kinase (5.6 units) was then added and thereaction continued for 30 min at 37° C. The fragments (except for the 5'terminii) were combined, diluted to 40 μl with water followed byaddition of 60 μl of 1M sodium acetate, 12 μl of 250 mM EDTA, and 5 μgof 1 mg/ml poly-dA. After heating for 5 min at 65° C., the 5' terminalpieces were added, followed by 420 μl of ethanol (100%). The solutionwas chilled for 20 min at -80° C., centrifuged, and the pellet waswashed twice with ethanol (100%) and dried. The pellet was redissolvedin water (18 μl), heated to 100° C. for 2 minutes and then cooled slowlyover 1.5 hours to 25° C. in a water bath.

The annealed fragment pool was ligated in a reaction mixture containingT4 DNA ligase (New England Biolabs, 1200 units) 1 mM ATP, 10 mM DTT, 10mM MgCl₂, 100 ng/ml spermidine, and 50 mM Tris-HCl, pH 7.8 (30 μl).After 1 hour at 14° C., the reaction mixture was partially purified bypreparative polyacrylamide gel (7%, native) electrophoresis. The DNA wasremoved from the appropriate gel slice by electroelution and ethanolcoprecipitation with poly dA (5 μg).

After assembly, the synthetic gD-A and gD-B fragments were substitutedinto a KpnI/SalI digested bacterial cloning plasmid pAB114αEGF-24, whichplasmid was prepared by cloning a mutagenized fragment of pYEGF-8 intopAB114 (see FIG. 1). The plasmids resulting from the insertion weredesignated pAB114αHS109 (gD-A) and pAB114αHS110 (gD-B).

The preparation of pAB114 was as follows: Plasmid pAB101 was obtainedfrom the screening of a random yeast genomic library cloned in YEp24(Fasiolo et al., 1981, J. Biol. Chem. 256: 2324) using a synthetic20-mer oligonucleotide probe (5'-TTAGTACATTGGTTGGCCGG-3') homologous tothe published α-factor coding region (Kurjan and Herskowitz, Abstracts1981, Cold Springs Harbor meeting on the Molecular Biology of Yeasts,page 242). Plasmid AB11 was obtained by deleting the HindIII to SalIregion of pBR322. An EcoRI fragment of pAB101 carrying the α-factor genewas then inserted into the unique EcoRI site pAB11 to produce pAB112.Plasmid pAB112 was digested to completion with HindIII, and thenreligated at low (4 μg/ml) DNA concentration to produce plasmid pAB113in which three 63 bp HindIII fragments were deleted from the α-factorstructural gene, leaving only a single copy of mature α-factor codingregion. A BamHI site was added to plasmid pAB11 by cleavage with EcoRI,filling in of the overhanging ends by the Klenow fragment of DNApolymerase, ligation of BamHI linkers and religation to obtain a plasmiddesignated pAB12. Plasmid pAB113 was digested with EcoRI, theoverhanging ends filled in, and ligated to BamHI linkers. Afterdigestion with BamHI, the resulting 1500 bp which carries the singlecopy of the α-factor gene fragment was gel-purified and ligated to pAB12which had been digested with BamHI and treated with alkaline phosphataseto produce pAB114, which contains a 1500 bp BamHI fragment carrying theα-factor gene.

The preparation of pYEGF-8 was as follows: A synthetic sequence forhuman epidermal growth factor (EGF) was prepared and ligated to pAB112(described above) which had been previously completely digested withHindIII and SalI to produce pAB201. The HindIII site lies within the3'-end of the α-factor gene, and the EGF sequence was inserted usingappropriate linkers. The resulting plasmid was designated pAB201.

Plasmid pAB201 (5 μg) was digested to completion with EcoRI and theresulting fragments were filled in with DNA polymerase I Klenow fragmentand ligated to an excess of BamHI linkers. The resulting 1.75 kbpfragment was isolated by preparative gel electrophoresis, andapproximately 100 ng of this fragment was ligated to 10 ng of yeastplasmid pC1/1 (described below) which had been previously digested tocompletion with restriction enzyme BamHI and treated with alkalinephosphatase. The ligation mixture of the 1.75 kbp fragment carrying thepartial α-factor gene fused to the EGF gene and pC1/1 was used totransform E. coli HB101 cells, and transformants were selected based onampicillin resistance. DNA from one ampicillin resistant clone(designated pYEGF-8) was used to transform yeast AB103 (genotype: MATα.pep 4-3, leu 2-3, lue 2-112, ura 3-52, his 4-580, cir°) cells, andtransformants selected based on their leu⁺ phenotype.

Plasmid pC1/1 is a derivative of pJDB219 (Beggs (1978) Nature 275: 104)where the region derived from bacterial plasmid pMB9 has been replacedby pBR322. The pC1/1 plasmid carries genes for both ampicillinresistance and leucine prototrophy.

Plasmid pAB114αEGF-24 was generated by an in vitro mutagenesis procedurewhich deleted the sequences coding for the glu-ala processing region inthe α-factor leader. Plasmid pAB114αEGF-24 was obtained as follows: aPstI-SalI fragment of pYEGF-8 containing the α-factor leader hEGF fusionwas cloned in phage M13 and isolated in single-stranded form. Asynthetic 36-mer oligonucleotide primer(5'-GGGGTACCTTTGGATAAAAGAAACTCCGACTCCGAA-3') was used as a primer forthe synthesis of the second strand using the Klenow fragment of DNApolymerase I. After fill-in and ligation at 14° C. for 18 hours, themixture was treated with S₁ nuclease and used to transfect E. coli JM101cells. Phage containing DNA sequences in which the glu-ala region wasremoved were located using ³² P-labelled primer as a probe. DNA frompositive plaques was isolated, digested with PstI and SalI, and theresulting fragment inserted into pAB114 (described above) which has beenpreviously digested to completion with SalI, partially with PstI andtreated with alkaline phosphatase. The resulting plasmid was designatedpAB114αEGF-24.

Referring again to FIG. 1, the BamHI-BamHI fragment of pAB114αHS109 orpAB114αHS110 (1588 base pairs for gD-A and 1546 base pairs for gD-B) wasexcised and ligated into the unique BamHI site of pC1/1. The resultingexpression vectors were designated pYHS109 for gD-A and pYHS110 forgD-B.

II. Expression of gD-A and gD-B polypeptides.

Plasmids pYHS109 and pYHS110 were both used to transform yeast strainAB103.1 (α, pep 4-3, leu 2-3, leu 2-112, ura 3-52, his 4-580, cir°) toleu prototrophy following the procedure of Hinnen et al. (1978) Proc.Natl. Acad. Sci. USA 75: 1929-1933. The transformants were grown in 1 Lcultures at 30° C. in buffered leucine-dificient media to saturation,corresponding to an absorbance of 5 at 650 nm. Yeast cell cultures weremaintained at saturation for an additional 12 to 24 hrs with shaking at30° C. The cultures were then harvested, the intact yeast cells pelletedby centrifugation at 3000 RPM, and the resulting supernatant mediafiltered through a 0.22μ Millipore filter. This fraction was then passedthrough a C18 reverse phase column, constructed from 8 Seppak unitspurchased from Waters. The bound material was eluted with 30 ml of 80%(v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid in water, evaporatedto dryness with a Buchii RotoVap and redissolved in 1.6 ml of distilledwater. This material was separated on a HPLC C18 column monitored at 210nm. The peak corresponding to each respective peptide was collected andits identity confirmed by antigenicity. Each peptide reactedspecifically in an ELISA assay using rabbit polyclonal antisera whichhad been raised against a chemically synthesized gD-B peptide or partialgD-A peptide (residues 256 through 271 of sequence shown in page 9)purchased from Vega Biochemicals. Expression levels, as determined byspectrophotometric measurements of HPLC purified peptides, were on theorder of 7.6 mg of gD-A per liter of yeast culture (OD₆₅₀ =5) and 0.6 mgof gD-B per liter of yeast culture (OD₆₅₀ =5). These results demonstratethe feasibility of expressing a relatively short portion or fragment ofa protein and its secretion from yeast cells using an α-factorexpression vector.

III. Construction of yeast vectors for high level intracellularexpression using fragments of the naturally-occurring gD gene

Nucleotide fragments from the naturally-occurring gD gene expressing gDof HSV-1 (gD-1) were also expressed intracellularly in yeast undercontrol of the GAPDH promoter and terminator. A library of EcoRIfragments of HSV-1, strain Patton, cloned into the EcoRI site of pBR322,was made by Dr. Richard Hyman, Hershey Medical Center, Hershey, Pa. ThegD is entirely contained with a 2.9 kb SacI fragment within the EcoRIfragment of one clone (clone H) isolated from the HSV-1 library. Clone Hwas obtained from Dr. Hyman, the 2.9 kb fragment was purified by gelelectrophoresis and was used for the construction of several expressionvectors which differ in the size of the gD fragment cloned and/or thesynthetic linkers used in the 5' or 3' ends of the gD fragments. FIG. 2illustrates the protein coding region (boxed region) and the fragmentsused for the construction of yeast expression vectors pYHS115, pYHS116,pYHS117, pYHS118 and pYHS119. A description of the construction of eachplasmid follows.

1. Construction of pYHS115

Plasmid pYHS115 contains the gD gene in a glyceraldehyde-3-phosphatedehydrogenase (GAPDH) expression cassette cloned into the BamHI site ofpC1/1 (described hereinabove).

The GAPDH expression cassette was constructed as follows. Threefragments were prepared (as described in detail below):

(a) A BamHI-HindIII fragment (1407 bp) containing 346 bp of pBR322 and1061 bp of the GAPDH promoter;

(b) A HindIII-SalI fragment (1430 bp) containing the gD gene, and

(c) A SalI-BamHI fragment (900 bp) containing the GAPDH terminator.

These fragments were ligated together and the mixture was digested withBamHI. The 3.7 Kb resulting cassette was isolated by gel electrophoresisand ligated to BamHI cut, alkaline phosphatase-treated pC1/1 (FIG. 3).

Fragment (a) was prepared by completely digesting pGAP347 (describedbelow) with BamHI followed by partial digestion with HindIII. Theresulting 1407 bp fragment containing 346 bp of pBR322 and 1061 bp ofthe GAPDH promoter was isolated by gel electrophoresis.

Construction of pGAP347 was as follows (see copending application Ser.No. 468,589, filed Feb. 22, 1983):

PolyA+RNA was isolated from S. cerevisiae yeast strain A364A.Double-stranded cDNA was synthesized using AMV reverse transcriptase andE. coli DNA polymerase I. Poly-dC-tails were added to thedouble-stranded cDNA molecule using deoxynucleotide terminaltransferase. Poly-dC-tailed cDNA was annealed to poly-dG-tailed pBR322and used to transform E. coli HB101. 1000 transformants were screened bycolony hybridization to labeled PolyA+RNA, and a subset further examinedby restriction endonuclease mapping, and DNA sequencing. Three clonescontaining GAPDH sequences were isolated from the pool. One clone(pcGAP-9) contained an insert of about 1200 base pairs and was used forfurther work.

A yeast gene library was prepared by inserting fragments obtained afterpartial digestion of total yeast DNA with restriction endonuclease Sau3Ain lambda phage Charon 28, according to Blattner et al. (1977) Science196: 161-169. Several fragments containing yeast GAPDH coding sequenceswere isolated by screening the phage library with labeled DNA frompcGAP-9. The yeast GAPDH gene of one of these clones was subcloned inpBR322 as a 2.1 kb HindIII fragment (pGAP-1). The GAPDH promoter regionwas isolated from these clones. A HhaI-HindIII fragment of about 350 bpcontaining the 3' portion of the promoter was obtained by: (a) digestionof pGAP-1 with HinfI to generate an approximately 500 bp segment whichincludes the 3' part of the promoter and a region encoding theN-terminal amino acids of GAPDH; (b) resection with Bal31 to yield a 400bp fragment lacking the GAPDH coding region (3'-terminus one baseupstream from the ATG initiator codon); (c) addition of HindIII linkers;and (d) cleavage with HhaI. A second HindIII-HhaI fragment of about 700bp containing the 5' portion of the promoter was isolated from pGAP-1,ligated to the 350 bp HhaI-HindIII fragment and treated with HindIII.The resulting 1061 bp HindIII fragment was isolated by gelelectrophoresis and cloned in HindIII digested, alkaline phosphatasetreated pBR322 to produce pGAP347.

Fragment (b) was obtained as follows. Clone H, isolated from the HSV-1Patton library was digested with SacI. A 2.9 Kb SacI fragment waspurified by gel electrophoresis and subsequently digested with HindIIIand NruI. The 1430 bp HindIII-NruI fragment containing the gD gene (FIG.3) was purified by gel electrophoresis, ligated to NruI-SalI adaptors ofthe following sequence: ##STR3## and digested with SalI.

Fragment (c) was obtained as follows. A 900 bp fragment containing theGAPDH terminator was obtained by BamHI and SalI digestion of pUH28(described under "Construction of pYHS117") and purification by gelelectrophoresis.

2. Construction of pYHS116

pYHS116 contains a gD gene fragment which has a 600 bp deletion at the5' end of the coding region that comprises most of the signal sequencecoding region. To construct pYHS116, two fragments were obtained:

(a) A BamHI-HindIII fragment (1407 bp) containing 346 bp of pBR322 and1061 bp of the GAPDH promoter. This fragment was obtained as describedunder "Construction of pYHS115."

(b) A NcoI-BamHI fragment (2150 bp) containing the partial gD genefollowed by the GAPDH terminator. This fragment was obtained byBamHI/NcoI digestion of pYHS115 (described previously) and purificationby gel electrophoresis. A HindIII-NcoI chemically synthesized adaptor ofthe following sequence ##STR4## was ligated to the fragment. Thisadaptor provides for the first two codons (met and val) fused in thecorrect reading frame to the partial gD.

Fragments (a) and (b) were ligated together and subsequently digestedwith BamHI. The resulting 3.5 Kb cassette was isolated by gelelectrophoresis and ligated to BamHI cut, alkaline phosphatase treatedpC1/1.

3. Construction of pYHS117

Plasmid pYHS117 contains the same partial gD gene clone in pYHS116,fused in reading frame to 7 extra codons at the 5'-end, which code forthe first 7 amino acids of the GAPDH structural gene. To constructpYHS117 two fragments were obtained.

(a) An NcoI-SalI digested vector (6.8 Kb) comprising pBR322 sequences,the GAPDH promoter fused to the first 7 codons of the structural geneand the GAPDH terminator. This vector was prepared by NcoI digestion ofpUH28 (described below), followed by a partial digestion with SalI andpurification by gel electrophoresis.

(b) An NcoI-SalI fragment (1430 bp) containing a partial gD gene. Thisfragment was obtained by BamHI-SalI digestion of pYHS115 (describedpreviously in Section II.1.) and purification by gel electrophoresis.

These two fragments were ligated together to yield a pBR322 derivedvector which contains a partial gD gene fused in reading frame to the 7first codons of GAPDH gene, flanked by the GAPDH promoter in its 5' endand by the GAPDH terminator in its 3' end. The gD expression cassettewas obtained by digesting this plasmid with BamHI and purifying a 3.4 Kbfragment by gel electrophoresis. This fragment was ligated to BamHIdigested, alkaline phosphatase treated pC1/1 to produce pYHS117.

Plasmid pUH28 contains the coding and 3' non-coding regions of thehepatitis B surface antigen (HBsAg) gene fused in incorrect readingframe to the first 7 codons of the GAPDH structural gene. This fusion isflanked in its 5' end by the GAPDH promoter and in its 3' end by part ofthe GAPDH coding region followed by the GAPDH terminator. This plasmidwas constructed so as to have an NcoI site at the 3' end of the first 7codons of the GAPDH gene with the following sequence: ##STR5## When thisNcoI end is ligated to the partial gD fragment (b, described above) thecorrect reading frame for the gD protein is regenerated. The SalI siteused in the preparation of fragment a (described above) is at the 5'region of the GAPDH terminator. Therefore, a deletion of the sAg codingplus non-coding regions and GAPDH coding region was obtained bydigesting pUH28 with NcoI and partially with SalI.

The construction of pUH28 involves cloning of a fragment that containsthe HBsAg coding and 607 bp of 3' non-coding region prepared frompHBS5-3 Hae2-1 (described below) into the GAPDH containing vector pGAP₂' (described below). To prepare the fragment, pHBS5-3 Hae2-1 waslinearized by PstI digestion, partially digested with NcoI and aPstI-NcoI fragment of 1.9 Kb containing pBR322 sequences, HBsAg codingand 3' sequences was purified by gel electrophoresis. This fragment wassubsequently digested with EcoRI and a 1.2 Kb NcoI-EcoRI fragmentcontaining the HBsAg coding and 3' non-coding regions was purified bygel electrophoresis. Plasmid pGAP₂ ' was linearized with XbaI andtreated with Bal31 to remove approximately 100 bp. The plasmid wassubsequently digested with NcoI and a vector fragment of about 9 Kb waspurified by gel electrophoresis. The NcoI ends of the vector and the 1.2Kb NcoI-EcoRI fragment encoding HBsAg were ligated. The recessed end wasfilled in with Klenow and the resulting blunt end was ligated to theblunt end of the vector obtained by Bal31 digestion to produce pUH28.

pHBS5-3 Hae2-1 is a plasmid that contains the HBsAg coding region and607 bp of 3' flanking sequences. This plasmid is a derivative of pHBS5-3which contains the same insert but only 128 bp of 3' untranslated regioninstead of 607 bp. Plasmid pHBS5-3 has been previously described incopending application, Ser. No. 609,540, filed May 11, 1984, (pp.13-14). pHBS5-3 Hae2-1 was constructed as follows. The HBV genome (3.2kb) was excised from pHB-3200 (Valenzuela et al., 1979, Nature, 280:815-819) by restriction digestion with EcoRI. The 3.2 kb fragment waspurified by gel electrophoresis and was recircularized by ligation ofthe EcoRI sticky ends. This closed HBV genome was digested with HaeII,which cuts in the 3' non-coding region. Recessed ends were filled inwith Klenow and HindIII linkers were ligated. The DNA was cut withHindIII and subsequently with XbaI, which has a single site in the HBScoding region. A 1.2 kb XbaI-HindIII fragment containing 586 base pairsof the coding sequence of HBV and 607 base pairs of the 3' non-codingregion was isolated by gel electrophoresis. This fragment was clonedinto pHBS5-3 previously cut with XbaI and HindIII and treated withalkaline phosphatase, to yield pHBS5-3 Hae2-1.

pGAP-2 is a pBR322 derived vector which contains a BamHI insert that hasthe GAPDH coding sequence, 5' and 3' flanking regions. There are twoXbaI sites in this plasmid: one in the coding region and one in the 3'flanking sequences. pGAP-2' is a derivative of pGAP-2 in which the XbaIsite present in the 3' flanking region has been eliminated. For thispurpose, 50 μg of pGAP-2 were partially digested with XbaI, treated withBal31 to remove 25 base pairs per end, and ligated. The plasmids wereused to transform E. coli HB101 and the transformants were selected forloss of the XbaI site in the 3' flanking region.

4. Construction of pYHS118

This vector contains a partial gD gene with deletions in two regions: a600 bp deletion in the 5'-end coding region which comprises most of thesignal sequence coding region and a 1300 bp deletion in the 3'-endcoding region which includes most of the anchor sequence coding region.It also contains 7 extra codons from the GAPDH gene coding region fusedin reading frame at the 5' end of the gD gene, similar to pYHS117.Plasmid pYHS118 was constructed as follows. pYHS115 was digested withNcoI and SalI, the resulting 1430 bp fragment containing the partial gDwas purified by gel electrophoresis and submitted to digestion with NarI(FIG. 2). The two resulting fragments (fragment a: 873 bp containing 5'end and fragment b: 411 bp containing 3' end) were independentlyisolated by gel electrophoresis. Fragment b was subsequently digestedwith Sau96A to yield three fragments which were separated by gelelectrophoresis. The 87 bp Nar-Sau96A fragment was recovered from thegel and was ligated to Sau96I-SalI synthetic adaptors of the followingsequence: ##STR6## The NarI-(Sau96A)SalI fragment (102 bp) was digestedwith SalI, purified by gel electrophoresis and ligated with fragment a(previously described). The resulting NcoI-SalI fragment (975 bp) wasligated to NcoI-SalI digested and gel purified pUH28 as described under"Construction of pYHS117." The resulting pBR322 derived vector wasdigested with BamHI and a 3.1 Kb fragment containing the gD expressioncassette was purified by gel electrophoresis. This cassette was ligatedto BamHI digested, alkaline phosphatase treated pC1/1 to producepYHS118.

5. Construction of pYHS119

This vector contains a partial gD gene with deletions in two regions: a600 bp deletion in the 5' end coding region which comprises most of thesignal sequence coding region and a 2400 bp deletion in the 3' endcoding region which includes all the anchor sequence coding region andabout 700 bp upstream of the anchor sequence. It also contains 7 extracodons from the GAPDH gene coding region fused in reading frame at the5' end of the gD gene, as pYHS117 and pYHS118. Plasmid pYHS119 wasconstructed as follows. pYHS115 was digested with NcoI and SalI, theresulting 1430 bp fragment containing the partial gD was purified by gelelectrophoresis and subsequently digested with NarI. The 873 bpNcoI-NarI fragment was isolated by gel electrophoresis. A syntheticadaptor of the following sequence: ##STR7## which provides complementarynucleotides to the NarI 5' overhang, 3 codons in reading frame, a stopcodon and a 5' overhang of SalI, was ligated to the 873 bp NcoI-NarIfragment then digested with SalI. The resulting NcoI-SalI fragment wasligated to pUH28 which has been previously completely digested with NcoIand partially digested with SalI and purified by gel electrophoresis asdescribed under "Construction of pYHS117." The resulting pBR322 derivedvector was digested with BamHI and a 2.2 Kb fragment containing the gDexpression cassette was purified by gel electrophoresis. This cassettewas ligated to BamHI digested, alkaline phosphatase treated pC1/1 toproduce pYHS119.

IV. Synthesis of gD from vectors containing partial or complete gD gene

Plasmids pYHS115, 116, 117, 118 and 119 were used to transform yeaststrain AB103.1 (α, pep 4-3, leu 2-3, leu 2-113, ura 3-52, his 4-580,cir°) following the procedure of Hinnen et al., (supra.). Thetransformants were grown to an OD₆₅₀ =3 at 30° in YEPD media. Thecultures were then harvested by pelleting the yeast cells at 3000 RPM.Cells were spheroplasted with zymolyase and subsequently osmoticallylysed in a hypotonic solution. Membranes were spun down in an eppendorfcentrifuge, and the pellet was solubilized in 0.1% SDS with proteaseinhibitors for 16 hours at 4° C. The suspension was centrifuged andtotal protein, as well as gD specific protein, was determined in bothsoluble and insoluble fractions. Expression of the gD gene in each ofthe above described constructions was detected by Western Blothybridization (Towbin et al., 1979, Proc. Natl. Acad. Sci. USA 76:4350).For this purpose protein samples were submitted to SDS-polyacrylamidegel electrophoresis (Laemmli (1970) Nature 227:680) and electroblottedonto nitrocellulose filters (Towbin et al. supra.). The filter waspreincubated with goat serum and subsequently treated with a rabbitpolyclonal antibody raised against HSV-1 (Dako). The filter was thenincubated with a second goat anti-rabbit antibody conjugated withhorseradish peroxidase (Boehringer-Mannheim) and finally it wasincubated with horseradish peroxide color development reagent (Bio-Rad)and washed. The results indicate that immunoreactive material is beingsynthesized in yeast AB103.1 strain transformed with the gD expressionvectors, with the exception of transformants containing pYHS115. In allother cases, gD protein corresponds to 0.1 to 0.5% of total yeast cellprotein.

According to the present invention, novel DNA constructs are providedfor expressing high levels of a polypeptide which is immunologicallycross-reactive with herpes simplex virus glycoprotein D. Such expressionis carried out in yeast. The genes expressed may be natural, syntheticor a combination of both, and secretion facilitates recovery of the gDgene product. Expression of both short, synthetic fragmentscorresponding to a portion of the gD protein and long, natural fragmentscorresponding to the entire gD protein has been demonstrated. Bothapproaches yield polypeptides that are immunologically reactive withantisera raised against natural gD protein.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. An improved method for producing a polypeptidewhich is immunologically cross-reactive with glycoprotein D of herpessimplex virus, said method comprising:growing a yeast host which hasbeen modified to include a DNA sequence expressing a polypeptide of atleast nine amino acids defining at least one epitopic site found innaturally-occurring glycoprotein D; and collecting the polypeptideproduced by the yeast host.
 2. A method as in claim 1, wherein the DNAsequence is under the transcriptional and translational control ofregulatory sequences including a secretory leader and processing signalsequence recognized by the yeast host.
 3. A method as in claim 1,wherein the DNA sequence is expressed intracellularly.
 4. A method as inclaim 1, wherein the DNA sequence is under the transcriptional controlof a promoter derived from a gene in the yeast glycolytic pathway.
 5. Amethod as in claim 4, wherein the DNA sequence is under thetranscriptional control of a promoter derived from a yeastglyceraldehyde phosphate dehydrogenase gene.
 6. A method as in claim 1,wherein the yeast host was modified by transformation with a DNAconstruct capable of self-replication in yeast, said DNA constructcarrying the glycoprotein D DNA sequence joined at its 5'-end totranscriptional and translational initiation regulatory sequences and atits 3'-end to transcriptional and translational termination regulatorysequences.
 7. A method as in claim 6, wherein the transcriptional andtranslational initiation regulatory sequences include a secretory leaderand processing signal sequence derived from yeast α-factor.
 8. A methodas in claim 1, wherein the polypeptide encoded by the DNA sequenceincludes amino acids 8 through 23 of naturally-occurring glycoprotein D.9. A method as in claim 1, wherein the polypeptide encoded by the DNAsequence includes amino acids 253 through 283 of naturally-occurringglycoprotein D.
 10. A method as in claim 1, wherein the DNA sequence isderived from a HindIII-NruI fragment of a glycoprotein D gene of herpessimplex virus type
 1. 11. A method as in claim 1, wherein the DNAsequence is derived from a NcoI-NruI fragment of a glycoprotein D geneof herpes simplex virus type
 1. 12. A method as in claim 1, wherein theDNA sequence is derived from a NcoI-Sau96A fragment of a glycoprotein Dgene of herpes simplex virus type
 1. 13. A method as in claim 1, whereinthe DNA sequence is derived from a NcoI-NarI fragment of a glycoproteinD gene of herpes simplex virus type
 1. 14. A method as in claim 9,wherein the DNA sequence is:5'-TTGCCACCAGAATTGTCTGAAACCCCAAACGCTACCCAAAACGGTGGTCTTAACAGACTTTGGGGTTTGCGATGGGTTCCAGAATTGGCTCCAGAAGACCCAGAAGACTCTGCTTTGTTGGAAGACCCA-3'GGTCTTAACCGAGGTCTTCTGGGTCTTCTGAGACGAAACAACCTTCTGGGT.
 15. A method as inclaim 8, wherein the DNA sequenceis:5'-TCTTTGAAGATGGCTGACCCAAACAGATTCAGAGGTAAGGACTTGCCA-''AGAAACTTCTACCGACTGGGTTTGTCTAAGTCTCCATTCCTGAACGGT.
 16. A DNA constructcapable of expression in a yeast host of a polypeptide which isimmunologically cross-reactive with glycoprotein D of herpes simplexvirus, said construct comprising:a DNA sequence encoding at least ninecontiguous amino acids defining at least one epitopic site found innaturally-occurring glycoprotein D; transcriptional and translationalinitiation regulatory sequences joined to the 5'-end of the DNAsequence, which initiation sequences are recognized by yeast;transcriptional and translational termination regulatory sequencesjoined to the 3'-end of the DNA sequence, which termination sequencesare recognized by yeast; and a yeast replication system.
 17. A DNAconstruct as in claim 16, wherein regulatory sequences include asecretory leader and processing signal sequence.
 18. A DNA construct asin claim 16, wherein the transcriptional initiation sequences include apromoter derived from a gene in the yeast glycolytic pathway.
 19. A DNAconstruct as in claim 18, wherein the promoter is derived from a yeastglyceraldehyde phosphate dehydrogenase gene.
 20. A DNA construct as inclaim 16, wherein the DNA sequence is a synthetic fragment encoding anepitope that is cross-reactive between types 1 and 2 of glycoprotein D.21. A DNA construct as in claim 20, wherein the epitope will elicitneutralizing antibodies to both types 1 and
 2. 22. A DNA construct as inclaim 21, wherein the DNA sequence encodes for at least nine amino acidsoccupying positions 8 through 23 of naturally-occurring glycoprotein D,employing preferred yeast codons.
 23. A DNA construct as in claim 21,wherein the DNA sequence encodes for at least nine amino acids occupyingpositions 253 through 283 of naturally-occurring glycoprotein D,employing preferred yeast codons.
 24. A DNA construct as in claim 16,wherein the DNA sequence is derived from a NcoI-NruI fragment of aglycoprotein D gene of herpes simplex virus type
 1. 25. A DNA constructas in claim 16, wherein the DNA sequence is derived from a NcoI-Sau96Afragment of a glycoprotein D gene of herpes simplex virus type
 1. 26. ADNA construct as in claim 16, wherein the DNA sequence is derived from aHindIII-NruI fragment of a glycoprotein D gene of herpes simplex virustype
 1. 27. A DNA construct as in claim 16, wherein the DNA sequence isderived from a NcoI-NarI fragment of a glycoprotein D gene of herpessimplex virus type
 1. 28. A DNA construct as in claim 22, wherein theDNA sequence is:5'-TCTTTGAAGATGGCTGACCCAAACAGATTCAGAGGTAAGGACTTGCCA-3'AGAAACTTCTACCGACTGGGTTTGTCTAAGTCTCCATTCCTGAACGGT.
 29. A DNA construct asin claim 23, wherein the DNA sequenceis:5'-TTGCCACCAGAATTGTCTGAAACCCCAAACGCTACCCAAAACGGTGGTCTTAACAGACTTTGGGGTTTGCGATGGGTTCCAGAATTGGCTCCAGAAGACCCAGAAGACTCTGCTTTGTTGGAAGACCCA-3'GGTCTTAACCGAGGTCTTCTGGGTCTTCTGAGACGAAACAACCTTCTGGGT.
 30. A plasmidselected from the group consisting of pHS109, pHS110, pYHS115, pYHS116,pYHS117, pYHS118, and pYHS119.